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ItemNew insights regarding the myocardial-adipose paracrine axis( 2021)Background: Pericardial adipose accumulation is a major risk factor for atrial fibrillation, independent of body mass index and non-cardiac adipose tissue volumes. Pericardial adipose is associated with detrimental perturbations in atrial electrophysiology, however the cellular mechanisms behind this relationship are poorly understood. Investigations into the pericardial adipose-myocardium paracrine axis have focused on pro-inflammatory/fibrotic factors, without assessing the direct paracrine influence of pericardial adipose on atrial electrophysiology. It is hypothesised that atrial electrophysiology is selectively impaired by pericardial adipose secreted factors compared to non-cardiac adipose, due to differences in the types of secreted proteins. Research Aims: (Relevant chapters in brackets) 1. Optimise and compare electrophysiology recordings of different types of immortalised and primary cardiomyocyte monolayers. (2) 2. Assess whether secreted factors specific to pericardial adipose can influence cardiomyocyte electrical conduction properties. (3) 3. Determine whether greater cardiac adiposity alters pericardial adipose paracrine phenotype and its influence on cardiomyocyte function. (4) 4. Compare and contrast protein release from sheep epicardial, paracardial and subcutaneous adipose tissue samples, to evaluate paracardial adipose as a potential paracrine mediator of cardiac pathology. (5) Methods: In vitro electrophysiology experiments (multi-electrode array) were optimised with neonatal rat ventricular myocytes (NRVM) and compared to immortalised atrial HL-1 cultures. To assess the paracrine influence of pericardial adipose, ovine and murine adipose tissue was incubated to produce a ‘conditioned media’. Multi-electrode mapping of HL-1 cultures was performed in the presence of pericardial adipose conditioned media from normal weight vs obese mice, and murine pericardial vs subcutaneous adipose. Identification of adipose secreted proteins was achieved by explorative proteomics using LC-MS/MS instrumentation. Subsequent gene ontology analysis (Enrichr and PINE software) was applied to proteomic datasets to profile differences between protein subgroups secreted from subcutaneous and pericardial adipose in mice and sheep. Results: Some of the major findings include: 1. Multi-electrode electrophysiological recordings could be comprehensively analysed with HL-1 and NRVM cultures. HL-1 cultures had a slower conduction velocity and shorter field potential repolarisation yet were less variable than NRVMs. 2. In obesity, murine pericardial adipose secreted proteins were substantially different to those secreted from subcutaneous adipose. Slowed electrical propagation was observed in HL-1 cell monolayers that received pericardial adipose conditioned media only. 3. The paracrine influence of pericardial adipose on HL-1 cell electrophysiology, or the types of pericardial adipose secreted proteins, are not altered by cardiac adiposity. Extracellular vesicle and focal adhesion associated proteins were identified in pericardial secretome from both normal and obese mice. 4. Ovine paracardial and epicardial adipose secreted proteins had a high amount of commonality, which included proteins related to inflammation, focal adhesion, and extracellular vesicles. The few points of contrast involved low-density lipoproteins, which may have implications in coronary artery disease. Conclusions: Atrial electrical propagation is selectively slowed by pericardial adipose through a direct paracrine action on cardiomyocytes. The types of proteins secreted by pericardial adipose were highly similar in samples from normal weight mice, obese mice, and normal weight sheep, yet contrasted significantly to subcutaneous secreted factors. The evidence provided herein collectively indicates that the association between obesity and atrial fibrillation is defined by the extent of cardiac adipose accumulation and therefore paracrine potential, and less so by pericardial adipose secretome profile. Therapeutic interventions which limit the release of conduction modulating adipokines from pericardial adipose tissue may provide a novel preventative treatment strategy for re-entrant arrhythmias.
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ItemPericardial adiposity and cardiac arrhythmia vulnerability( 2020)Background: The cellular mechanisms that predispose to arrhythmia include heterogeneic conduction slowing and/or changes in repolarisation time (e.g. regional alterations in transmural electrophysiology). Augmented pericardial adiposity is an independent risk factor for atrial and ventricular fibrillation, but the cellular mechanisms are unknown. Very limited data indicate pericardial adipose tissue exhibits paracrine characteristics, secreting factors which modulate cardiac electrophysiology. Recent evidence demonstrates pericardial adipose tissue can synthesise oestrogens which are known to affect cardiomyocyte function. It is hypothesised that augmented pericardial adiposity promotes epicardial conduction slowing and/or repolarisation prolongation through paracrine mechanisms to predispose to arrhythmia. Research aims: (relevant chapters in brackets) 1. Establish that transmural electrophysiology is modulated in the context of elevated cardiac adiposity. (3) 2. Compare electrophysiology of cardiomyocyte cultures from different origins as prelude to examining the paracrine influences of pericardial adipose tissue. (4) 3. Ascertain that obesity and epicardial adiposity associate with cardiac electrophysiological remodelling which may increase arrhythmia vulnerability. (5) 4. Identify that sex steroids can modulate atrial electrophysiology, indicating their potential as paracrine regulators of arrhythmia vulnerability. (6) Methods: Cardiac electrophysiology was assessed in both atrial and ventricular tissues utilising multiple in vitro methodologies. To establish the effects of adiposity on transmural electrophysiology, male rats were fed a high fat diet, then tangential left ventricular slices were electrophysiologically mapped. To optimise cardiomyocyte culture conditions, neonatal rat ventricular myocyte (NRVM) and human induced pluripotent stem cell-derived cardiomyocyte (hiPSC-CM) electrophysiology was compared. Fragments of epicardial adipose tissue were co-cultured with hiPSC-CMs to assess the paracrine influence on cardiomyocyte electrophysiology. The effects of obesity on left atrial electrophysiology were determined using male mice fed a Western diet. Left atrial electrophysiology was also assessed in male and female chow-fed mice in the absence/presence of sex steroids. Results: Some of the overall findings of this investigation include: 1. Augmented pericardial adiposity likely disrupts ventricular transmural conduction gradients through putative local actions on the epicardium. 2. hiPSC-CM and NRVM cultures display similar electrophysiology and exhibit good capacity to detect changes in repolarisation via experimental intervention. 3. Obesity associates with prolonged epicardial atrial action potential duration. This is caused by a paracrine influence of pericardial adipose tissue on cardiomyocytes. 4. Oestrogen and testosterone prolong repolarisation and slow conduction in the left atrium, indicating their potential as paracrine regulators of arrhythmia vulnerability. Conclusions: Pericardial adipose tissue has capacity to selectively prolong epicardial activation and repolarisation. This is at least in part, mediated through a paracrine mechanism. Prolonged repolarisation and slowed conduction predispose to triggered and reentrant arrhythmias, respectively. Together, these data indicate that augmented cardiac adiposity has a causative effect on cardiomyocyte electrophysiology to increase arrhythmia likelihood.